Macrophage numbers in the marginal area of sarcomas predict clinical prognosis

Even when treated comprehensively by surgery, chemotherapy, and radiotherapy, soft-tissue sarcoma has an unfavorable outcome. Because soft-tissue sarcoma is rare, it is the subject of fewer clinicopathological studies, which are important for clarifying pathophysiology. Here, we examined tumor-associated macrophages in the intratumoral and marginal areas of sarcomas to increase our knowledge about the pathophysiology. Seventy-five sarcoma specimens (not limited to a single histological type), resected at our institution, were collected, and the number of CD68-, CD163-, and CD204-positive macrophages in the intratumoral and marginal areas was counted. We then performed statistical analysis to examine links between macrophage numbers, clinical factors, and outcomes. A high number of macrophages positive for all markers in both areas was associated with worse disease-free survival (DFS). Next, we divided cases according to the FNCLCC classification (Grade 1 and Grades 2/3). In the Grade 1 group, there was no significant association between macrophage number and DFS. However, in the Grade 2/3 group, high numbers of CD163- and CD204-positive macrophages in the marginal area were associated with poor DFS. By contrast, there was no significant difference between the groups with respect to high or low numbers of CD68-, CD163-, or CD204-positive macrophages in the intratumoral area. Multivariate analysis identified the number of CD163- and CD204-positive macrophages in the marginal area as an independent prognostic factor. Macrophage numbers in the marginal area of soft-tissue sarcoma may better reflect clinical behavior.

IHC evaluation. All IHC slides were scanned at an absolute magnification of 20 × using a pathology digital imaging system (Nanozoomer virtual slide system, Hamamatsu Photonics, Shizuoka, Japan). For each slide, five representative fields (0.2 mm 2 ) containing intratumoral and marginal areas were selected, and the number of positive cells was counted manually (Fig. 1a). The selected intratumoral and marginal areas were almost same for the CD68, CD163, and CD204-stained slides. The marginal area is the non-sarcoma area adjacent to the sarcoma. For most sarcomas, the histological findings identified a non-sarcomatous area bordering the sarcomatous area, which was designated as the "marginal area". In cases where the border between sarcoma and non-sarcoma was unclear, we carefully distinguished atypical cells from preexisting tissue, and designated the non-sarcoma area around the sarcoma margin as the "marginal area". The total number of CD68-, CD163-, or CD204-positive cells in the five tumor fields was recorded (Fig. 1b,c). There were two cases in which the marginal specimen did not include normal tissue; in these cases, the marginal tumor area was used instead of the marginal normal area. Both cases involved an atypical lipomatous tumor.
Statistical analysis. Shapiro-Wilk test showed the number of macrophages are not normal distribution, therefore, the non-parametric analyses described below were performed. The number, distribution, and type of macrophage was assessed using the Friedmann rank sum test with Bonferroni correction, and the Wilcoxon signed rank test ( Table 2). The correlation between the number of intratumoral/marginal macrophages of each phenotype was assessed using Spearman's rank correlation analysis (Fig. 2). Clinical factors and macrophage type were assessed using the Mann-Whitney U test, the Kruskal-Wallis test, and the Steel-Dwass test ( Patient consent status. Informed consent was obtained in the form of opt-out on the website.

Results
Distribution of each macrophage phenotype, and the correlation between intratumoral and marginal macrophages of each phenotype in soft-tissue sarcoma. The number of CD68-, CD163-, and CD204-positive macrophages in the intratumoral and marginal areas of 75 soft-tissue sarcoma specimens was counted. A comparison of macrophage numbers in the intratumoral and marginal areas revealed that the number expressing each of these markers was significantly higher in the intratumoral area than in the marginal area (CD68-, CD163-, and CD204-positive macrophages: p = 0.00000109, p = 0.000013, and p = 0.00000711, respectively) ( Table 2). For each particular marker, there was an intermediate correlation between the numbers in the marginal and intratumoral areas (CD68-positive macrophages: r = 0.575; CD163-positive macrophages: r = 0.644; CD204-positive macrophages: r = 0.618; p < 0.001 for all) (Fig. 2a-c). By contrast, a comparison of the number of macrophages expressing each type of marker revealed that the number of CD163-positive macrophages in both areas was significantly lower than that of CD68-and CD204positive macrophages (intratumoral area: p = 0.000233; marginal area: p = 0.00000283) ( Table 2). In both areas, there was an intermediate-to-strong correlation between the number of CD68-, CD163-, and CD204-positive macrophages in each examined area (intratumoral CD68-and intratumoral CD163-positive macrophages: r = 0.907; intratumoral CD68-and intratumoral CD204-positive macrophages: r = 0.876; intratumoral CD163and CD204-positive macrophages; r = 0.86; marginal CD68-and CD163-positive macrophages: r = 0.697; marginal CD68-and CD204-macrophages: r = 0.804; marginal CD163-and CD204-positive macrophages; r = 0.72; p < 0.001 for all) (Fig. 2d-i).
Association between the number of CD68-, CD163-, and CD204-positive macrophages in each area and clinical factors. The number of each type of marker-positive macrophage in both areas of the tumor, and their association with clinical factors, was analyzed statistically (Table 3). Sex, age, tumor size, or location had no effect on the type of marker-positive macrophage either area. However, there were significant     Association between the number of macrophages in the intratumoral and marginal areas and DFS. Next, we performed a Kaplan-Meier analysis of the groups with a high and low number of CD68-, CD163-, and CD204-positive macrophages in both tumor areas. All cases with high macrophage counts in both areas, showed significantly worse DFS than those with low counts (intratumoral CD68-, CD163-, and CD204-positive macrophages: p = 0.00595, p = 0.0178, and p = 0.00116, respectively; marginal CD68-, CD163-, and CD204-positive macrophages: p = 0.0108, p = 0.0000735, and p = 0.0000011, respectively) ( Fig. 4). Next, we divided the population into two subgroups (FNCLCC Grade 1 and FNCLCC Grades 2/3), and analyzed DFS. In the Grade 1 group, high numbers of CD68-and CD204-positive macrophages in both areas tended to be associated with worse DFS (intratumoral CD68-and CD204-positive macrophages: p = 0.189 and p = 0.362, respectively; marginal CD68-and CD204-positive macrophages: p = 0.252 and p = 0.64 respectively) (Fig. 5), although the results did not reach the threshold for significance. In the Grade 2/3 group, there was no significant difference between them with respect to the phenotype of macrophage in the intratumoral area; however, high numbers of CD163-and CD204-positive macrophages in the marginal area were associated with significantly worse        (Fig. 6). We also assessed metastasis-free survival in patients with Grade 2/3 sarcoma (Fig. 7). High numbers of CD68-, CD163-, or CD204-positive macrophages in the marginal area were associated with poorer metastasis-free survival (marginal CD68-, CD163-, and CD204-positive macrophages: p = 0.03, p = 0.0305, and p = 0.0254, respectively); however, the number of macrophages (of any phenotype) in the intratumoral area had no significant effect on metastasis-free survival in either group. Finally, we assessed local recurrence-free survival for those with Grade 2/3 sarcoma. Again, the number and phenotype of macrophages in the intratumoral or marginal areas had no significant effect (see Supplementary Fig. S1 online).

Prognostic value of the number of CD163-or CD204-positive macrophages in the marginal area.
We performed multivariate analysis to analyze the prognostic value of the number of marginal CD163and CD204-positive macrophages and other clinical factors (sex, age, location, size, and FNCLCC grade; see Table 4) for all cases. Multivariate analysis of DFS identified the numbers of marginal CD163-and CD204-positive macrophages as independent predictors of a poorer prognosis for Grades 1, 2, and 3 (marginal CD163-and CD204-positive macrophages: p = 0.04739 and p = 0.003541, respectively).

Discussion
Compared with cancer in general, sarcoma is quite rare. Furthermore, soft-tissue sarcoma includes many histological types; therefore, it is difficult to collect cases limited to a specific single histological type for statistical analysis. For these reasons, few studies have performed clinicopathological examination of TAMs in sarcoma.
Here, we also reviewed previous studies that investigated the association between TAMs and clinical outcomes of patients with soft-tissue sarcoma (Table 5) 7,[30][31][32][33][34] . Most previous studies report that high macrophage counts are associated with poor outcomes; however, some do not show a significant association between TAMs and clinical outcome. Also, none of these studies evaluated macrophages in the marginal area, as we did here. We collected cases regardless of histology; however, we excluded cases that had undergone preoperative chemotherapy or radiotherapy. In the overall population, the macrophage count (all phenotypes) in both areas showed a significant association with DFS. These results suggest that the number of macrophages in the intratumoral or marginal area is associated with the clinical grade of sarcoma. This is supported by our finding that the number of macrophages, especially CD163-or CD204-positive macrophages, in the intratumoral or marginal area was associated with FNCLCC grade (Fig. 3). In the Grade 1 subgroup, high numbers of CD68-and CD204-positive macrophages tended to be associated with a poorer prognosis, although there was no significant difference between macrophage numbers in the two areas. We think that if the number of cases had been larger, we may have found a significant difference. Interestingly, we found that patients in the Grade 2/3 subgroup with high numbers of CD163-and CD204-positive macrophages in the marginal area showed significantly poorer DFS; this association was not present when we examined the intratumoral area. The finding that high numbers of intratumoral macrophages (CD68-positive, CD163-positive, or CD204-positive) tended to be associated with somewhat poorer DFS is consistent with previous studies. To evaluate DFS, we defined local recurrence and metastasis as a clinical event. We considered that local recurrence depends somewhat upon the surgical method (marginal or extensive resection); thus, we also assessed metastasis-free survival in those with Grade 2/3 sarcoma. We found that those with high numbers of macrophages in the marginal area showed poorer outcomes (as was observed for DFS). Multivariate analysis identified a high number of CD163-or CD204-positive macrophages in the marginal area as an independent prognostic factor.
We suggest three explanations for the above results. One involves the types of cases that were enrolled in the study. Unlike previous studies, our cases were of various histological type. The pathological characteristics and clinical behavior of sarcomas differ among histological types, which may be one reason for the differences in the results. Second, the accuracy of the macrophage counts should be considered. In cases of high-grade sarcoma, especially undifferentiated sarcoma, the sarcoma cells themselves are sometimes CD68-positive. Actually, almost all cases diagnosed as undifferentiated pleomorphic sarcoma today may have been previously diagnosed as malignant fibrous histiocytoma. We did not find an sarcoma cells that were CD163-and CD204-positive; however, the possibility that some were CD163-or CD204-positive cannot be ruled out (indeed, a case of CD163-positive sarcoma has been reported 35 ). In addition, high-grade sarcomas often show dense infiltration of the intratumoral area by CD68-/CD163-/CD204-positive cells, as demonstrated in Fig. 1c. These findings make precise counting of CD68-, CD163-, and CD204-positive cells very difficult; thus, the number of macrophages in the intratumoral area may not be precise. In turn, the number of macrophages in the marginal area, which was lower than that in the intratumoral area, was easier to count because the macrophages were easier to distinguish from sarcoma cells. Of course, quantification of the macrophages in the marginal area was also associated with some problems. One such problem occurs in cases involving marginal resection. Some cases, especially low-grade sarcoma (e.g., atypical lipomatous tumors) are treated by marginal resection. In this study, we carefully analyzed small amounts of the non-sarcoma (i.e., normal) area adjacent to the sarcoma and counted the number of macrophages in those areas. However, in cases in which we could not find a marginal normal area, the marginal tumor area was investigated instead. Another problem is that there were cases with an unclear demarcation line. In such cases, we carefully chose the area to analyze, making sure not to count sarcoma cells. Despite these problems, for Grade 2/3 sarcomas, the number of the macrophages in the marginal area show a stronger correlation with prognosis than the number in the intratumoral area. Lastly, marginal macrophages may play an important role in sarcoma progression. As we described earlier, macrophages play an important role in formation of the tumor microenvironment in gastric cancer 28 . If this is the same for sarcoma, then the number of macrophages in the marginal area may reflect the clinicopathological behavior of the sarcoma.
Biomolecular studies are needed to confirm the hypothesis that macrophages in the marginal area play an important role in progression of sarcoma. Similar to other studies, the results of the present study suggest that there are therapeutic benefits in targeting macrophages as a treatment for sarcoma 36 .

Conclusion
We investigated the numbers of CD68-, CD163-, and CD204-positive macrophages in the intratumoral and marginal areas of sarcoma cases. In all cases (Grade 1/2/3), we found that patients with high numbers of intratumoral macrophages (CD68 + , CD163 + , and CD204 +) and high numbers of marginal macrophages (CD68 + , CD163 + , and CD204 +) showed poor DFS. In Grade 2/3 cases, those with high numbers of CD163-and CD204-positive macrophages in the marginal area showed a poorer prognosis than those with low numbers, whereas there was no significant difference in prognosis of those with high and low numbers of CD68-, CD163-, or CD204-positive macrophages in the intratumoral area.

Data availability
All data generated or analyzed during this study are included in the published article.

Funding
The research was funded by JSPS Kakenhi Grants (Number 21K15380 to MU).